转vp28基因工程聚球藻的规模培养与采收

发布时间：2018-05-09 08:42

本文选题：聚球藻 + 光生物反应器　；参考：《上海海洋大学》2016年硕士论文

【摘要】：对虾白斑综合症病毒(WSSV)于1992年首次在台湾发现,随后迅速蔓延至各大洲,对全世界的对虾养殖业造成严重的病害,经济损失超过70亿美元。国际上,尚不存在产业化的WSSV防治药物。研究发现,WSSV被膜中的特异性结构蛋白VP28在病毒侵染细胞过程中发挥关键作用,该蛋白经异源系统表达后,通过口服或注射接种,可提高对虾抗WSSV的能力。我们的研究也发现,转vp28基因工程蓝藻经对虾口服后攻毒,发现对虾在30天内能有效抵抗WSSV(成活率达70%-90%),表明已开发的工程聚球藻具有巨大的产业化前景。目前本实验室转vp28基因工程聚球藻口服剂的研制已进入实验准备阶段,其中工程聚球藻的规模培养及采收是其产业化的重要一环。我们在进行工程聚球藻规模培养中,认识到其产业化受3方面因素制约:1)目前规模培养设备及技术不成熟,生物量产量较低;2)重组蛋白表达率尚有提高的潜力;3)缺少安全快速高效的采收技术。为解决上述问题,本论文特从以下3方面进行系统研究:1.光照强度对工程聚球藻生物量的调控为提高转vp28基因工程聚球藻的生物量产量,光照强度(I)的调控依据2个因素:1)I与p H、温度的交互作用;2)I在反应器中的衰减作用。为此,本论文首先通过单因素实验,确定了当净光合速率为极大值时,I、温度及p H的交互范围,分别为200-400μmol/m2s、28-48°C、6.5-8.5;并进一步通过响应面实验测定I、p H及温度的最优交互条件,即分别为300±10μmol/m2s、37°C、7.5,此时净光合速率达到最大值,342μmol O2/mg Chlah。按照优化温度(37°C)和pH(7.5),分别于2种反应器模型(5 L/108 W外置光源模型,100 L/120W内置光源模型)中培养工程聚球藻。5 L反应器中I在培养对数期可调至250-300μmol/m2s,3 d后生物量为1.5 g/L;100 L反应器中I在培养对数期可调至180-250μmol/m2s,7 d后生物量为0.4 g/L。对100 L反应器进行改造,将灯管功率由120 W提高至300 W,同时将光源分布由3组改为6组,培养至对数期时,反应器50%区域中I达到300±10μmol/m2s,并使对数期延长36 h,7 d后生物量达到0.8 g/L。可见,I是影响光生物反应器最终生物量的关键因素,提高I的功率能够显著增加生物量产量,使I均匀分布可显著延长生长对数期。2.光质对工程聚球藻重组蛋白VP28表达的影响通过改变光质可以调控工程聚球藻的生长及重组蛋白的表达。采用5 L/108 W外置光源反应器进行72 h培养,通过改变入射光中单色光的比例,检测培养过程中生物量的变化、psb A的转录、vp28的转录及VP28蛋白的相对积累量。研究发现:1)光质为50%白光、33.3%蓝光及16.7%红光时,最终vp28的表达率达到2.4%,是100%白光下的3倍;重组蛋白VP28的相对积累量提高至2倍;生物量的积累达到1.22 g/L;psb AII、psb AIII的转录得到促进。2)光质为50%白光、33.3%红光及16.7%蓝光时,最终vp28的表达率达到0.5%;10μg总蛋白中VP28的相对积累量只有高比例蓝光下的5%;psb AII及psb AIII基因的转录受到抑制,psb AI的转录得到促进;生物量积累达到1.68g/L。可见,光质可显著调控重组蛋白的表达,并对生物量产生一定影响。其中蓝光促进了重组蛋白的表达,抑制了生物量的积累,而红光与蓝光的作用相反。光质对质粒启动子的调控机制与宿主基因psb A的调控有关。3.工程聚球藻采收技术的研究为建立快速安全高效的采收技术,研究不溶性沉淀絮凝法的采收效率及安全性,主要包括3个方面:1)最适诱导剂及其剂量的确定;2)细胞悬浮液中胞外分泌物(AOM)的去除;3)絮凝后蛋白析出量控制。实验结果显示,1)在悬浮液中增加0.25 m M镁离子浓度,在p H 11条件下采收率达到90%以上。2)4000 rpm离心去除悬浮液中的AOM,采用新鲜培养液(0.3 m M镁离子)重悬,发现p H调至10.5时,采收率即达到95%以上。3)发现光质为50%白光、33.3%蓝光及16.7%红光时,悬浮液中AOM为2 mg/L以下。4)100%白光下,悬浮液AOM为4.5 mg/L左右。5)光质50%白光、33.3%红光及16.7%蓝光下,悬浮液中AOM为5 mg/L以上。6)在5°C、20°C及35°C水浴条件下,分别检测絮凝结束后上清液中的总蛋白含量,30 min后,分别为35μg/ml、24μg/ml、10μg/ml。其中,可溶性蛋白经电泳分离后,分子量大小均在20 KDa以下,经抗体检测未发现VP28条带。7)絮凝后及时调整下层藻细胞浓缩液至p H 7.5,上清液蛋白析出量为12μg/ml。可见,镁离子的絮凝效果受到AOM的严重制约,高比例蓝光可显著降低AOM的产生,高比例红光起相反效果。采收后藻细胞的蛋白损失受温度及p H影响,避免高温并及时恢复p H至藻细胞生理水平,可降低50%蛋白损失。综上我们认为,应用光生物反应器培养转vp28基因工程聚球藻时,光照强度及光质的合理调控能显著提高生物量及重组蛋白产率;改良的不溶性沉淀絮凝法可在不损伤重组蛋白的条件下,实现了工程聚球藻的快速采收。
[Abstract]:The shrimp white spot syndrome virus (WSSV) was first discovered in Taiwan in 1992, and then spread rapidly to various continents, causing serious diseases to the whole world's prawn breeding industry, and the economic loss was more than 7 billion dollars. Internationally, there is no industrial WSSV control drug. The study found that the specific structural protein VP28 in the WSSV membrane was infected by the virus. The cell process plays a key role in increasing the ability of the shrimp to resist WSSV by oral or injection inoculation. Our study also found that the transgenic VP28 gene engineering cyanobacteria could effectively resist WSSV (the survival rate of 70%-90%) in 30 days, indicating the developed engineering poly ball. Algae have great industrialization prospects. At present, the development of VP28 gene engineering polychlorella oral agent in our laboratory has entered the experimental preparation stage. The scale culture and collection of Engineering polychlorella is an important part of its industrialization. We recognize that the industrialization of polychlorella is restricted by 3 factors: 1) in the scale culture of polychlorella engineering. The former scale culture equipment and technology are not mature, the biomass yield is low; 2) the potential of recombinant protein expression is still improved; 3) lack of safe and rapid and efficient harvesting technology. In order to solve the above problems, this paper is to systematically study the following 3 aspects: 1. the regulation of the biomass of polychlorobacteria by 1. light intensity is to improve the transformation of VP28 gene engineering. The biomass yield of alga and the regulation of light intensity (I) are based on 2 factors: 1) the interaction between I and P H, temperature, and 2) the attenuation effect of I in the reactor. For this reason, this paper first determined the interaction range of I, temperature and P H when the net photosynthetic rate was maximum, and the interaction range of I, temperature and P H, respectively, was 200-400, 28-48 [28-48] C, and 6.5-8.5; and The optimal interaction conditions of I, P H and temperature were measured by the response surface experiment, which were 300 + 10 mol/m2s, 37, C, 7.5, at this time the net photosynthetic rate reached the maximum, 342 Mu mol O2/mg Chlah. in accordance with the optimum temperature (37 degrees C) and pH (7.5), respectively in 2 reactor models (5 L/108 W external light source model, 100 built-in light source model). In the.5 L reactor of Engineering polychlorella, the I in the logarithmic period of culture can be adjusted to 250-300 mu mol/m2s, and the biomass is 1.5 g/L after 3 D; I in the 100 L reactor can be adjusted to 180-250 micron in the logarithmic period of culture, and the biomass of 7 d is 0.4 g/L. to 100 L reactor, and the lamp power is raised from 120 to 300, and the distribution of the light source is changed from 3 to 6. When cultured to logarithmic phase, I in the 50% region of the reactor reached 300 + 10 mu mol/m2s, and the logarithmic period was extended by 36 h, and the biomass reached 0.8 g/L. after 7 d. I was the key factor affecting the final biomass of the bioreactor. The increase of the power of I could significantly increase the biomass yield, and the uniform distribution of I could significantly prolong the logarithmic phase of the growth of.2. light. The expression of polychlorella recombinant protein VP28 could regulate the growth of polychlorella and the expression of recombinant protein by changing light quality. The 5 L/108 W external light source reactor was used for 72 h culture. By changing the proportion of monochromatic light in the incident light, the change of biomass, the transcription of PSB A, the transcription of VP28 and the VP28 protein were detected. The study found that: 1) when the light quality is 50% white light, 33.3% blue light and 16.7% red light, the final expression rate of VP28 reaches 2.4%, 3 times in 100% white light; the relative accumulation of recombinant protein VP28 increases to 2 times; the accumulation of biomass reaches 1.22 g/L; PSB AII, PSB AIII is transcribed to promote.2) to be 50% white light, 33.3% red light and 16.7%. In blue light, the expression rate of the final VP28 reached 0.5%; the relative accumulation of VP28 in the total protein of 10 mu g was only 5% under the high proportion of blue light; the transcription of PSB AII and PSB AIII genes was inhibited, the transcription of PSB AI was promoted; the biomass accumulation reached 1.68g/L., and the light quality could significantly regulate the expression of the recombinant protein and had a certain effect on the biomass. Blue light promotes the expression of recombinant protein and inhibits the accumulation of biomass, but the effect of red light and blue light is opposite. The study of the regulation mechanism of the plasmids promoter and the regulation of the host gene PSB A related to the.3. engineering polychlorella harvesting technology is to establish a fast and safe and efficient harvesting technology and study the extraction of insoluble precipitation flocculation method. The efficiency and safety include 3 aspects: 1) the determination of the optimum inducer and its dosage; 2) the removal of extracellular secretions (AOM) in cell suspension; 3) control of protein precipitation after flocculation. Experimental results showed that 1) increased the concentration of 0.25 m M magnesium ions in the suspension, and the recovery rate of more than 90%.2 under the condition of P H 11) and 4000 rpm centrifugation AOM in the suspension was suspended with fresh medium (0.3 m M magnesium ions), and when p H was adjusted to 10.5, the recovery rate was above 95%.3) and the light quality was 50% white, 33.3% blue light and 16.7% red light, AOM in the suspension was 2 mg/L below.4) 100% white, the suspension AOM was 4.5 mg/L.5), 50% white light, 33.3% red light and 16.7% blue light. The total protein content in the supernatant after flocculation was detected under the conditions of 5 degree C, 20 degree C and 35 degree C in the floating solution, and the total protein content in the supernatant after flocculation was detected respectively. After 30 min, the total protein content was 35 mu g/ml, 24 g/ml and 10 micron g/ml. respectively. After the soluble protein was separated by electrophoresis, the molecular weight was below 20 KDa. After the antibody detection did not detect the flocculation of the VP28 strips, it was timely after flocculation. To adjust the concentration of lower algae cell concentrate to P H 7.5, the amount of protein precipitation in the supernatant is 12 u g/ml., the flocculation effect of magnesium ions is seriously restricted by AOM, and the high proportion of blue light can significantly reduce the production of AOM. The high proportion of red light plays the opposite effect. The protein loss of the algae cells after harvesting is affected by the temperature and P H, avoiding the high temperature and recovering the H to algae in time. The level of cell physiology can reduce the loss of 50% protein. In the conclusion that the light intensity and light quality control can significantly increase the biomass and the yield of recombinant protein when using the photo bioreactor to train VP28 genetically engineered polychlorella, and the improved insoluble precipitation flocculation can achieve the engineering polymerization without damaging the recombinant protein. The fast harvest of the alga.

【学位授予单位】：上海海洋大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：Q949.2

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